Nucleic acid hybridization assays are commonly used in genetic research, biomedical research and clinical diagnostics. In a basic nucleic acid hybridization assay, single-stranded analyte nucleic acid is hybridized to a labeled single-stranded nucleic acid probe and resulting labeled duplexes are detected. Variations of this basic scheme have been developed to enhance accuracy, facilitate the separation of the duplexes to be detected from extraneous materials, and/or amplify the signal that is detected.
The present invention is directed to a method of reducing background signals encountered in solution phase sandwich hybridization assays that derive from several sources. Generally, the background noise which is addressed by way of the presently disclosed techniques results from undesirable interaction of various polynucleotide components that are used in a given assay, i.e., interaction which gives rise to a signal which does not correspond to the presence or quantity of analyte. The invention is useful in conjunction with any number of assay formats wherein multiple hybridization steps are carried out to produce a detectable signal which correlates with the presence or quantity of a polynucleotide analyte.
One such assay is described in detail in commonly assigned U.S. Pat. No. 4,868,105 to Urdea et al. That assay involves the use of a two-part capturing system designed to bind the polynucleotide analyte to a solid support, and a two-part labeling system designed to bind a detectable label to the polynucleotide analyte to be detected or quantitated. The two-part capture system involves the use of capture probes bound to a solid support and capture extender molecules which hybridize both to a segment of the capture probes and to a segment of the polynucleotide analyte. The two-part labelling system involves the use of label probe extender molecules which hybridize to a segment of the polynucleotide analyte, and label probes which hybridize to the label probe extender molecules and contain or bind to a detectable label. An advantage of such a system is that a plurality of hybridization steps must occur in order for label to be detected in a manner that correlates with the presence of the analyte, insofar as two distinct hybridization reactions must occur for analyte "capture," and, similarly, two distinct hybridization reactions must occur for analyte labelling. However, there remain a number of ways in which a detectable signal can be generated in a manner which does not correspond to the presence or quantity of analyte, and these will be discussed in detail below.
Another example of an assay with which the present invention is useful is a signal amplification method which is described in commonly assigned U.S. Pat. No. 5,124,246 to Urdea et al. In that method, the signal is amplified through the use of amplification multimers, polynucleotides which are constructed so as to contain a first segment that hybridizes specifically to the analyte nucleic acid or a strand of nucleic acid bound to the analyte, and a multiplicity of second segments that hybridize specifically to a labeled probe. The degree of amplification is theoretically proportional to the number of iterations of the second segment. The multimers may be either linear or branched. Branched multimers may be in the shape of a fork or a comb, with comb-type multimers preferred.
One approach which has been proposed to increase the target dependence of the signal in a hybridization assay is described in European Patent Publication No. 70,685, inventors M. J. Heller et al. That reference describes a homogeneous hybridization assay in which a nonradiative transfer of energy occurs between proximal probes; two distinct events must occur for a target-generated signal to be produced, enhancing the accuracy of detection. A second approach designed to enhance the signal deriving from the presence of analyte is described in European Patent Publication No. 361,983 inventor J. E. Stefano. The method described therein involves hybridization of two probe sequences (one a midivariant RNA (MDV), the other a half-ribozyme), each of which is complementary to sequences present in an RNA target. The ribozyme thus formed specifically cleaves the tail of the MDV probe, releasing the MDV probe from the support and enhancing its replication. Still a third approach to increase specificity in hybridization assays is described by Distefano et al., J. Am Chem. Soc. (1992) 114:11006-11007. That method involves the use of short regions of double-helix formation to enhance the stability of two short regions of triple-helix DNA.
European Patent Publication No. 552,931, inventors Hogan et al., describe a nucleic acid hybridization assay utilizing a probe system that detects regions of double-stranded DNA that only form in the presence of target sequence.
The present invention, which does not rely on the detection of the presence of double-stranded regions, is also designed to increase the accuracy of detection and quantitation of polynucleotide analytes in hybridization assays. The invention increases both the sensitivity and specificity of such assays, by reducing the incidence of signal generation that occurs in the absence of target, and does not involve a substantial increase in either time or cost relative to current assay configurations. In certain embodiments, the invention is also effective in compensating for the loss in signal that can result when background noise is reduced.